Jeff:LabNotes/Microbiome/2012-8-23: Difference between revisions
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>Jgole (Created page with "== ''Tagmentation test using Genomic DNA''' == *Test 4 samples, each with 25 ng genomic DNA starting template *Sample 1: PC in water *Sample 2: DNA is EtOH precipitated and e...") |
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[[File: 11- | [[File: 11-14-12 pcr.jpg|400px]] [[File: 11-14-12 gel1.jpg|400px]] |
Revision as of 21:11, 14 November 2012
Tagmentation test using Genomic DNA'
- Test 4 samples, each with 25 ng genomic DNA starting template
- Sample 1: PC in water
- Sample 2: DNA is EtOH precipitated and eluted in water
- Sample 3: DNA in New prep below w/o Pol I. ALS and NS premixed as to be neutral
- Sample 4: Pol I treated DNA using New prep
Pol 1 treatment
- Start with 1 uL DNA
- Add 1.5 uL ALS, 3min
- Put on ice and add 1.5 uL NS
New Prep
Template 4 uL 10x buffer 4 .7 uL dNTP .35 uL N6(unmodified) .35 uL water .4 uL Pol I .5 uL ---------------------------------- Total 7 uL
- 37 C for 30 min, 65C for 10 min
Tagmentation
DNA in water 7 uL HMW buffer 2 uL, (.6 uL for New prep samples for a total of 2 uL) water (for new prep only) 1.4 uL 1:50 diluted enzyme 1 uL ---------------------- Total 10 uL
- 55C for 5 min
Protease treatment
- Add 1 uL 1:100 diluted protease
- 10 min 50C, 20 min 70C
Klenow
- Add 1 uL exo- klenow and 1 uL dNTP to each
- 37C for 15 min, 75 for 20 min
1st PCR round
DNA 13 uL KAPA Robust mix 15 uL Orange .6 uL blue .6 uL 50x SYBR .24 uL -------------------------- Total 30 uL
- 95 C 1min (95 C 10s, 65C 15s, 72 C 45s)x8
Second round PCR
- Pause PCR program and add following to each
1st round PCR mix 30 uL KAPA Robust mix 35 uL Adapter 1 2 uL Adapter 2 Barcode 2 uL SYBR Green 1 .56 uL water 30.44 uL ---------------------- Total 100 uL
1 min 95C, (95 C 10s, 65C 15s, 72 C 45s)x3, (95 C 10s, 72C 1 min)x4, 72C 3min
Bead purfication
- Add 100 uL beads to each, mix, and let sit at RT for 8min
- Transfer tubes to magnetic rack and let sit for 5 min
- Remove supernatant
- 2x 80% EtOH wash
- Sit for 15 min
- Elute in 23 uL Qiagen buffer
Results
- There is a 1 cycle difference between PC and EtOH, implicating 1/2 the DNA is lost
- Even though the buffer for the Pol I reactions is the same as tagmentation buffer, tagmentation is very inefficient. ALS is prepared as diluted KOH only (no DTT or EDTA). Not sure what is happening. Only differences are K and CL ions from ALS/NS, and dNTP's/N6 primer from Pol I mix
- When the Pol I reaction was run, it is entirely inhibited meaning the enzymes are somehow competing (even though Pol I is killed)
- In any case, EtOH continues to be the best option, and I will only use this in the future